functionalization of cobalt porphyrin-phospholipid bilayers with...

Functionalization of cobalt porphyrin–phospholipidbilayers with his-tagged ligands and antigensShuai Shao1,2, Jumin Geng1, Hyun Ah Yi3, Shobhit Gogia2, Sriram Neelamegham2, Amy Jacobs3

and Jonathan F. Lovell1,2*

Methods to attach polypeptides to lipid bilayers are often indirect and ineffective, and can represent a substantialbottleneck in the formation of functionalized lipid-based materials. Although the polyhistidine tag (his-tag) has beentransformative in its simplicity and efficacy in binding to immobilized metals, the successful application of this approach hasbeen challenging in physiological settings. Here we show that lipid bilayers containing porphyrin–phospholipid conjugatesthat are chelated with cobalt, but not with other metals, can effectively capture his-tagged proteins and peptides. Thebinding follows a Co(II) to Co(III) transition and occurs within the sheltered hydrophobic bilayer, resulting in an essentiallyirreversible attachment in serum or in a million fold excess of competing imidazole. Using this approach we anchoredhoming peptides into the bilayer of preformed and cargo-loaded liposomes to enable tumour targeting without disruptingthe bilayer integrity. As a further demonstration, a synthetic protein fragment derived from the human immunodeficiencyvirus was bound to immunogenic liposomes for potent antibody generation for an otherwise non-antigenic peptide.

The development of targeted nanoparticles that nimbly guidepayloads to diseased tissues1 and the rational design ofpeptide antigens that induce the immune system to combat

diseases2 have been the focus of considerable research efforts. Onebottleneck in advancing these techniques stems from difficultiesin easily and reliably attaching peptides and proteins to largerscaffolds; targeted nanoparticles require effective ligands and theunconjugated peptides themselves are weakly immunogenic.Bioconjugate chemistry has provided a range of strategies3, butmost nanoparticulate conjugations suffer from limitations thatrelate to one or more of the following: (1) low conjugation yieldsand the necessitated purification steps; (2) incompatibility withbiological buffers, which makes labelling of intact nanoparticlesimpossible; (3) variable labelling sites and conjugated polypeptideconformations, which create an inhomogeneous particle populationwith varying degrees of function; (4) the necessity for complex andexogenous chemical approaches4.

Standard approaches for ligand attachment to aqueous nanopar-ticles make use of maleimides, succinimidyl esters and carboiimide-activated carboxylic acids5. These can covalently react with theamine and thiol groups of polypeptides. The use of maleimidelipids has been explored extensively for antibody-conjugatedimmunoliposomes6. Conjugation yields may reach as high as 90%from an overnight reaction, but subsequent quenching of the freemaleimide groups and additional purification is required7.Proteins may require a preparative step of thiolation and purifi-cation prior to conjugation8. Antibody orientation is a majorfactor that influences the conjugated antibody target-binding effi-cacy, but these approaches result in numerous antibody labellingsites and indiscriminate orientations9. Biorthogonal synthetic strat-egies, such as the click reaction, have recently been applied to pre-formed liposomes; however, these require the use of exogenouscatalysts and unconventional amino acids10.

Another approach suitable for smaller peptides that are lessprone to permanent denaturation in organic solvents is to conjugate

the peptides to a lipid anchor. The resulting lipopeptides canthen be incorporated along with the other lipids during theliposome-formation process. This approach has been used to gener-ate synthetic vaccines that induce antibody production againstotherwise non-immunogenic peptides11. However, their amphi-pathic character means that the lipopeptides are difficult to purify.It has also been shown that lipopeptides do not fully incorporateinto liposomes during the formation process, which results in aggre-gation12. These problems necessitate evolving strategies to work withliposomal lipopeptides, such as adding multiple lipidation sites13.

One alternative strategy of interest that could avoid these limit-ations is to adopt the non-covalent method used in immobilizedmetal ion affinity chromatography (IMAC)14. Polypeptides contain-ing a polyhistidine-tag (his-tag) that comprises a stretch of approxi-mately 6–10 consecutive histidine residues can be purified from acrude lysate using immobilized microparticles decorated with Ni,Zn or Co chelators, such as nitrilotriacetic acid (NTA). Thisapproach has changed the face of biotechnology and is commonpractice for the purification of recombinant proteins15,16. Ni-NTAconjugated phospholipids, which were described over 20 years agoand are commercially available, have also been used to bindhis-tagged proteins, but largely in the context of structural biologystudies17–19. Unfortunately, the chelation of his-tagged proteinswith various Ni-NTA lipids in nanoparticulate form is not stablein biological media such as serum20,21. Based on the suitability oflipids for use in vivo in the form of liposomes or as coatings forother nanoparticles22, stable lipid-based polyhistidine bindingcould be enabling for applications in biological settings.

Porphyrin–phospholipid (PoP) conjugates have recently beendeveloped that can self-assemble into biodegradable nanovesiclesfor multimodal imaging and phototherapy23–26. With four cyclicnitrogens, PoP conjugates also represent potent chelators forexactly the same transition metals as frequently used in IMAC. Inthis work, we demonstrate that a metallo-PoP bilayer specificallyformed from cobalt porphyrin–phospholipid (Co-PoP) can stably

1Department of Biomedical Engineering, University at Buffalo, State University of New York, Buffalo, New York 14260, USA. 2Department of Chemical andBiological Engineering, University at Buffalo, State University of New York. Buffalo, New York 14260, USA. 3Department of Microbiology and Immunology,University at Buffalo, State University of New York, Buffalo, New York 14260, USA. *e-mail: [email protected]

ARTICLESPUBLISHED ONLINE: 20 APRIL 2015 | DOI: 10.1038/NCHEM.2236

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bind his-tagged polypeptides with simple aqueous incubation(Fig. 1a). This represents a new binding model, with the polyhisti-dine buried in the membrane phase as the porphyrins themselvesform the hydrophobic portion of the bilayer and are not accessibleto the external aqueous environment (Fig. 1b). This leads to a morestable binding, allows for significantly simpler non-covalent post-labelling methods after the nanoparticle formation and eliminatesambiguity regarding ligand orientation.

Results and discussionHis-tagged protein binding to Co-PoP liposomes. A series ofsn-1-palmitoyl sn-2-pyropheophorbide phosphtatidylcholinechelates was generated with the transition metals Co, Cu, Zn, Niand Mn (Fig. 1c). PoP bilayers were then formed with 10 mol%metallo-PoP along with 85 mol% dioleoylphosphatidylcholine(DOPC) and 5 mol% polyethylene glycol-conjugateddistearoylphosphatidylethanolamine (PEG-lipid) via extrusioninto 100 nm liposomes. His-tagged protein binding to the PoPbilayers was assessed with a fluorescent protein reporter. Asshown in Fig. 2a, the system comprised a fusion protein made upof two linked fluorescent proteins, Cerulean (blue emission) andVenus (green emission). Owing to their linked proximity andspectral overlap, Cerulean serves as a Förster resonance energytransfer (FRET) donor for Venus, so that the Cerulean excitationresults in FRET emission from Venus. Cerulean was tagged at itsC terminus with a heptahistidine tag. However, if bound to a PoPbilayer, energy transfer from Cerulean is diverted to the bilayer

itself, which is absorbing in the Cerulean emission range and thuscompetes with the FRET to Venus. Also, as Venus is not directlyattached to the photonic bilayer, it is not completely quenchedon direct excitation, which enables tracking of the boundfusion protein.

A three-colour electrophoretic mobility shift assay (EMSA) wasdeveloped to assess reporter fusion protein binding to various PoPliposomes. Protein (2.5 µg) was incubated with 50 µg of variousPoP liposomes for 24 hours and then subjected to agarose gelelectrophoresis. As shown in the top image in Fig. 2b, when thePoP liposomes were imaged, only the free base (2H) liposomeswere readily visualized, along with, to a lesser degree, the Zn-PoPliposomes. This demonstrates that the metals have a quenchingeffect on the PoP liposome and confirms they were stably chelatedin the bilayer. As expected, the liposomes exhibited minimal electro-phoretic mobility because of their relatively large size. Next, thesame gel was imaged using Cerulean excitation and Venus emissionto probe for the inhibition of FRET, which would be indicative of thefusion protein binding to PoP liposomes. All the samples exhibitedthe same amount of FRET and migrated the same distance as thefree protein, with the exception of the protein incubated withCo-PoP liposomes, for which the FRET disappeared completely(middle image). To verify the presence of the protein, Venus wasdirectly excited and imaged. Only with the Co-PoP liposomes wasthe reported protein colocalized with the liposomes. Together,these images demonstrate that the protein bound quantitatively tothe Co-PoP liposomes. Solution-based studies confirmed this

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Figure 1 | His-tagged polypeptides bind and functionalize Co-PoP bilayers. a, Schematic that shows a peptide with a his-tag (green) binding to preformedCo-PoP liposomes in an aqueous solution. A simple aqueous incubation results in the insertion of the his-tag into the bilayer and stable binding. b, Insertionof a his-tagged polypeptide into a bilayer containing Co-PoP (red) along with a majority of conventional phospholipids (blue). Only a single leaflet of thebilayer is shown. The imidazole groups of the histidine residues coordinate with cobalt within the hydrophobic bilayer phase. c, Chemical structure of themetallo-PoPs used in this study.

ARTICLES NATURE CHEMISTRY DOI: 10.1038/NCHEM.2236

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finding (Fig. 2c). Of all the types of PoP liposomes examined, only theCo-PoP ones induced a dramatic decrease in the FRET efficiencybetween Cerulean and Venus, caused by the liposomal binding. Thebinding required approximately a day to complete fully, althoughthe time to achieve a 50% binding (t1/2) was just three hours. It waspreviously shown by molecular dynamics simulations of a freebase(2H) PoP (2H-PoP) bilayer that the centres of the porphyrins(where metal chelation would occur) are inaccessible to the aqueousphase that surrounds the bilayer25. Thus, this slow binding can beattributed to a his-tag that is partially obscured by the rest of theprotein and has to make its way into the sheltered hydrophobic bilayer.

Polyhistidine coordination with Co-PoP. The mechanism thatunderlies his-tag binding to immobilized metals involves metalcoordination with the nitrogenous imidazole groups of histidineresidues. The absence of his-tag binding to liposomes formed withNi(II), Cu(II), Zn(II) and Mn(II) PoP probably relates to an axial

ligand-binding affinity or to the coordination number within theporphyrin. For instance, it has been proposed that Ni(II) and Cu(II)porphyrin chelates can coordinate completely with the foursurrounding macrocyclic nitrogens without axial ligands27. For theZn(II) and Mn(III) porphyrins, the ligand binding strength isprobably insufficient to confer stable polyhistidine binding. Theinorganic study of cobalt coordination was first pioneered by Wernerover 100 years ago28. Co(III) complexes are of interest because theyare often kinetically inert to ligand release and Co(III)-NTA hasrecently attracted interest for binding his-tag proteins withexcellent stability29. Co(II) porphyrins, such as the originallysynthesized Co-PoP, readily oxidize to Co(III) in aqueous solutions30.Confining Co(III)-mediated his-tag binding within a shelteredbilayer is expected to enhance further the binding stability inbiological environments.

To determine the electronic state of the Co-PoP, its paramagnet-ism was assessed. As Co(II) is paramagnetic, but Co(III) porphyrins

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Figure 2 | His-tagged protein binding to Co(III)-PoP liposomes. a, A heptahistidine (hepta-his)-tagged fluorescence protein that comprises Cerulean (C)fused to Venus (V) reveals binding to the PoP bilayers. When C is excited, FRET occurs and V emits fluorescence (left), but this is inhibited when bound tothe PoP bilayer because of FRET competing with the photonic bilayer (middle). The C fluorescence can be probed directly even when the protein is bound tothe bilayer (right). b, Multispectral fluorescence images of a fusion protein electrophoretic mobility shift after incubation with the indicated metallo-PoPliposomes. Arrows indicate the position of the protein bound to Co-PoP liposomes. c, Binding kinetics of the fusion protein to the indicated metallo-PoPliposomes based on the loss of C to V FRET. d, NMR peak widths of the underlined proton of the vinyl group on Co-PoP demonstrate paramagneticbroadening of Co(II) in deuterated chloroform (CDCl3) but non-paramagnetic peaks of Co(III) after Co-PoP liposome formation in deuterated water.e, Reversal of his-tagged peptide binding to Co-PoP liposomes after the addition of 2 M sodium sulfite. Liposomes were formed with 10 mol% Co-PoP orNi-NTA phospholipid. All the graphs show the mean ± s.d. for n = 3. em, emission; ex, excitation.

NATURE CHEMISTRY DOI: 10.1038/NCHEM.2236 ARTICLES





are low spin and diamagnetic30, NMR was used to probe for peakbroadening induced by paramagnetic species. As shown inFig. 2d, based on the hydrogens of each carbon of the vinyl groupwithin the PoP, a wide peak broadening was observed only for theCo-PoP, and only in an organic solvent. When the Co-PoP wasformed into aqueous liposomes, the peaks narrowed, which indi-cates oxidation to diamagnetic Co(III) within the bilayer. To verifythis mechanism further, the reducing agent sodium sulfite wasadded to the Co-PoP liposomes after they were quantitativelybound to a fluorescently labelled his-tagged peptide. As shown inFig. 2e, 2 M sulfite induced peptide release from the Co-PoP lipo-somes. Liposomes were also formed with a commercially availableNi-NTA lipid. The his-tagged peptide did not bind as avidly tothe Ni-NTA liposomes. On the addition of sulfite to the system,no release of the peptide was observed, as expected with Ni(II),which cannot be reduced readily. Together, these data suggest thatCo-PoP transitions from Co(II) to Co(III) on forming Co-PoP lipo-somes and the polyhistidine imidazole groups coordinate in thebilayer with chelated Co(III) in the PoP.

Stable his-tag binding to Co-PoP liposomes. The fluorescencereporter protein was then used to compare the binding of his-tagged proteins to liposomes that incorporated either Co-PoP orNi-NTA lipid (Fig. 3a). The Ni-NTA liposomes included 10 mol%2H-PoP to enable protein-binding determination based on FRET.By EMSA, the protein migrated unimpeded when incubatedwithout liposomes or when incubated with 2H-PoP liposomes inboth the FRET channel and the protein channel. When incubatedwith Ni-NTA liposomes, migration of the protein was onlyslightly inhibited, which indicates that the protein binding did notwithstand the conditions of electrophoresis. That the FRETchannel was unquenched confirmed a lack of binding to theNi-NTA liposomes. In contrast, when incubated with the Co-PoPliposomes, the protein was bound stably, with the completedisappearance of the FRET channel and a decreasedelectrophoretic mobility that is consistent with the proteinremaining bound to liposomes.

For biomedical applications, an intractable obstacle of using anNi-NTA lipid is that it does not maintain stable his-tag binding inbiological media such as serum20,21. To examine whether liposomescould maintain binding in the presence of serum, fetal bovine serum(FBS) was added at a 1:1 volume ratio to a solution of liposomesthat had bound the his-tagged protein. As shown in Fig. 3b,Ni-NTA liposomes did not fully sequester all the protein, which isconsistent with the weak binding exhibited in the EMSA result.After the serum addition, all the binding was abrogated over a24 hour period. Under the same conditions Co-PoP liposomesstably sequestered the his-tagged reporter protein withoutsubstantial protein release.

As the histidine side chain comprises an imidazole group, animidazole competition assay was used to compare the Ni-NTAand Co-PoP liposome binding stability with his-tagged poly-peptides. As shown in Fig. 3c, Co-PoP liposomes maintained anover 75% binding to the reporter protein, even at concentrationsthat approached 1 M imidazole. This represents an approximate tenmillion fold imidazole excess over the 100 nM protein concentrationused in the binding study. In contrast, the Ni-NTA liposomesreleased over 90% of the his-tag in the presence of just 30 mMimidazole. The drastically stronger binding of the Co-PoP liposometo the his-tag may be attributed to at least two factors: the superiorstable chelation of Co(III) to imidazole groups and the protectedhydrophobic environment of the Co-PoP bilayer, which limitsaccess to competing external molecules.

Liposomes formed with the Ni-NTA lipid, the cobalt-chelatedCo-NTA-lipid and the Co-PoP could bind a fluorescent peptidein solution (Supplementary Fig. 1a). However, the binding of

Co-NTA and Ni-NTA was not maintained during gel-filtrationchromatography (Supplementary Fig. 1b). Liposomes formedwith Co-NTA and Ni-NTA, but not with Co-PoP, released thepeptide when incubated in serum (Supplementary Fig. 1c). Thisdemonstrates the significance of bilayer-confined polyhistidinebinding. We next examined whether or not Co-PoP was requiredfor a stable binding in serum, or whether a simple liposome-inserted cobalt porphyrin would be sufficient. After the initialbinding, incubation with serum caused the polypeptide tobecome displaced from the liposomes (Supplementary Fig. 2).This result is consistent with demonstrations that membrane-inserted porphyrins, but not PoPs, rapidly exchange with serum

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Figure 3 | Robust his-tagged protein binding to Co-PoP liposomes.a, Multispectral electrophoretic mobility shift images of the fluorescentreporter protein incubated with liposomes that contained the indicated lipid.The arrows point to the his-tagged protein bound to Co-PoP liposomes,which exhibit inhibited electrophoretic mobility because of the large size ofthe complex. 2H-PoP liposomes and Ni-NTA liposomes did not stably bindthe protein. b, Binding stability of the reporter protein bound to the indicatedliposomes after the addition of serum. c, Binding stability of the reporterprotein bound to the indicated liposomes with excess free imidazole. All thegraphs show the mean ± s.d. for n = 3.






components and exit the liposome25. Together these results pointto the essential role Co-PoP plays to stably bind his-taggedpolypeptides.

Peptide binding to Co-PoP liposomes. Peptide targeting hasattracted interest for use as disease and tissue-specific ‘zipcodes’31. The short RGD tripeptide, which is found in fibronectinand vibronectin, is a promising targeting ligand for its effectivebinding to the integrin αVβ3 expressed on tumour endothelialcells32,33. Co-PoP liposomes were examined to verify whether theycan be delivered to molecular receptors on target cells via a his-tagged ligand approach with the short linear amino-acidsequence GRGDSPKGAGAKG-HHHHHHH. Carboxyfluorescein(FAM) was labelled on the N terminus to enable the detection ofbinding to a PoP liposome via FRET. It has been shown thatlinear RGD peptides can be labelled with fluorophores withoutdisrupting integrin binding34. As shown in Fig. 4a, when thispeptide was incubated with various metallo-PoP liposomes, onlythe Co-PoP ones bound the peptide. Compared to proteinbinding, peptide binding was about five times faster. Presumably,the smaller size, faster molecular motion and decreased sterichindrance of the peptide enabled a more rapid interdigitationinto the bilayer to interact with and irreversibly bind the Co-PoP.Based on previous estimates that each ∼100 nm liposomecontains approximately 80,000 lipids7, this equates to 8,000Co-PoPs and 750 peptides per liposome. As each peptidecontained seven histidine residues, the ratio of Co-PoP tohistidine in the bilayer was 1:0.66. This represents an excess ofCo-PoP and, for conventional his-tag binding to Ni-NTA, of allthe residues in the his-tag, only the ith and i + 2 or i + 5 histidineresidues are believed to be involved in coordinating with themetal14. However, the porphyrin and polyhistidine densitywithin the Co-PoP bilayer is probably higher and therefore the

coordination mechanism may be different. Further work isrequired to determine the geometry and stoichiometry of his-tagbinding in the Co-PoP bilayer phase.

The effect of his-tag length on peptide binding to the Co-PoPliposome was examined. A series of N-terminus FAM-labelled pep-tides was synthesized with varying lengths of his-tag attached to theC terminus. As demonstrated in Fig. 4b, when the his-tag wasomitted from the peptide, no peptide binding was observed. Withtwo histidine residues, the binding was slow, with a binding t1/2 ofnearly ten hours. As the his-tag length increased, the bindingspeed increased rapidly. With six residues, which corresponds tothe common hexahistidine tag, the binding t1/2 was less than onehour. By increasing the his-tag length to ten residues, the bindingt1/2 decreased to 20 minutes.

Next, the lipid composition was varied to determine the effect ofmembrane fluidity on his-tag binding. Liposomes were formed with90 mol% of either distearoylphosphatidylcholine (DSPC), dimyris-toylphosphatidylcholine (DMPC) or DOPC along with 10 mol%Co-PoP. Alternatively, 50 mol% cholesterol was also incorporatedin the bilayer with a corresponding reduction in the amount ofstandard lipid used. DSPC forms rigid, gel-phase bilayers at roomtemperature, whereas DMPC and DOPC have lower transitiontemperatures and are in the liquid-crystal phase at room tempera-ture. Cholesterol occupies space in the bilayer and can have a mod-erating effect on the membrane fluidity. Interestingly, no majordifferences were observed in the peptide-binding rate to membranesof different compositions, with or without cholesterol, with bindingt1/2 values of under one hour (Fig. 4c) and the times required forbinding 90% of the peptide being between 10 and 12 hours(Supplementary Fig. 3). The peptide-binding process might be amultistep one so that, once the peptide begins insertion into thebilayer, cooperative effects of the polyhistidine are minimally impactedby lipid composition and fluidity. In the presence of 5 mgml–1

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Figure 4 | Binding of a short his-tagged RGD peptide to Co-PoP liposomes. a, Binding of a short linear RGD-his peptide labelled with FAM to metallo-PoPliposomes. Only the Co-PoP liposomes bound the peptide. b, Effect of the his-tag length on binding t1/2 to Co-PoP liposomes. No binding (NB) was observedfor the peptide that lacked a his-tag. Increasing the his-tag length resulted in a faster binding. c,d, Effect of liposome composition on binding t1/2 to Co-PoPliposomes of the indicated composition when incubated in PBS (c) or in 5 mg ml–1 BSA (d). In the presence of BSA, the binding was slower for liposomesthat lacked cholesterol. All the graphs show the mean ± s.d. for n = 3.






bovine serum albumin (BSA), dramatic differences between themembranes with and without cholesterol were observed (Fig. 4d).The slower binding in cholesterol-free liposomes was probablybecause of a greater interaction of BSA with the membrane thatinterfered with peptide binding. Binding t1/2 values were notreached with BSA at 50 mg ml–1 and serum completely inhibitedbinding (Supplementary Fig. 4).

Biotargeting of cargo-loaded liposomes. Given the bindingefficacy of his-tagged peptides to Co-PoP liposomes, the bilayerintegrity was assessed to determine whether peptide bindinginduces membrane destabilization. The aqueous core of liposomeswas loaded with the fluorophore sulforhodamine B, a water-solubledye, at self-quenching concentrations to probe for membranepermeabilization. As shown in Fig. 5a, dye-loaded liposomes didnot release a substantial amount of dye over the eight hour periodin which the peptide had fully bound to the liposomes. At 24hours, the Co-PoP liposomes with the peptide bound released lessthan 10% of the dye. Thus, his-tag insertion and binding issufficiently gentle and non-disruptive for the bilayer integrity andentrapped cargo to remain intact. The analogous palmitoylatedlipopeptide resulted in permeabilization of the cargo-loadedliposomes on incubation (Supplementary Fig. 5), which furtherdemonstrates the robustness of the his-tag approach.

Next, the liposomes were assessed for whether they could bind totheir molecular targets with the established cell-line pair of U87

glioblastoma cells (RGD binding) and MCF7 breast-cancer cells(RGD non-binding)35,36. After sulforhodamine B entrapment, lipo-somes were first incubated with the his-tagged RGD peptide andthen with both cell lines. Approximately 550 peptides were attachedto each liposome. Liposomal uptake was assessed by examining thefluorescence in the cells after washing and lysis (to remove anyeffects of cargo self-quenching). As shown in Fig. 5b, a high lipo-some uptake was observed in the U87 cells incubated with the tar-geted Co-PoP liposomes, whereas negligible binding occurred withthe MCF7 cells. As expected, without the RGD-targeting ligand nouptake occurred in either cell line. Inclusion of PEG-lipid in theliposome formulation resulted in liposomes that did not target toeither cell line. It is probable that the presence of the PEG had theeffect of obstructing the peptide, which is directly tethered to thebilayer surface. Binding was inhibited with the presence of excessfree RGD peptide, which confirms the targeting specificity of theapproach. Confocal microscopy substantiated these binding results(Fig. 5c). Although the FAM-labelled peptide was quenched bythe PoP liposomes, sufficient signal remained to verify thebinding of both the targeting peptide and the liposomal cargo.Both the cargo and the peptide were internalized and remainedcolocalized in the U87 cells. When the Co-PoP liposomes andtargeting peptide were admixed immediately prior to incubationwith the U87 cells, the targeting peptide itself bound to the U87cells but did not have time to attach to the liposomes, whichremained untargeted. The same result was observed for 2H-PoP

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Figure 5 | RGD-his targeting of cargo-loaded liposomes. a, Release of entrapped sulforhodamine B in preformed PoP liposomes. Minimal membranedestabilization occurred in the process of his-tagged peptide functionalization. b, Targeted uptake of liposomes loaded with sulforhodamine B in U87 cellsknown to express αVβ3 or MCF7 control cells. Cells were incubated in the indicated conditions and uptake was assessed by examining sulforhodamine Bfluorescence in cell lysates. c, Confocal micrographs that show the liposome uptake. The cells were incubated with the indicated liposome solutions for twohours, washed and imaged. All the images were acquired with the same settings. d, Biodistribution of sulforhodamine B entrapped in Co-PoP liposomes withor without the attachment of a his-tagged cyclic RGD-targeting peptide 45 minutes after intravenous injection into nude mice that bore subcutaneous U87tumours. All the graphs show means ± s.d. for n = 3. *No preincubation.






liposomes, which did not bind the peptide. Peptide binding wasmaintained when the liposomes were formed with only 1 mol%Co-PoP (Supplementary Fig. 6a) and the selective binding to theU87 cells was maintained (Supplementary Fig. 6b). Cargo-loadedliposomes that incubated a his-tagged cyclic RGD (cRGD) moietywere injected intravenously into nude mice that bore U87tumours. As shown in Fig. 5d, 45 minutes after the intravenousinjection the targeted liposomes accumulated in tumours with a2.5-fold avidity compared to that of the untargeted liposomes.These data show that Co-PoP liposomes can be loaded with cargoin the core of the liposome, be labelled with a his-tagged targetingpeptide without inducing cargo leakage and be directed to molecu-lar receptors expressed on cells that express specific surface proteinsin vitro and in vivo.

Liposomes represent only a subset of all the types of nanomater-ials used in biomedical applications37. Co-PoP was assessed as a gen-eralized surface coating with a selective adhesion for his-tags. Goldnanoparticles were used as a model nanoparticle because these areused in numerous biological applications38. Using an establishedprotocol to lipid-coat gold nanospheres39, a citrate-stabilized 60nm gold dispersion was used to hydrate a thin film of PoP lipid.On repeated centrifugation and resuspension, the citrate wasdisplaced, which caused the nanospheres to aggregate(Supplementary Fig. 7a). However, in the presence of the PoPlipid, the nanospheres became coated and remained dispersible.The PoP-coated nanospheres had a slightly larger hydrodynamicsize than the citrate-stabilized gold because of the bilayer coatingon the gold (Supplementary Fig. 7b). The presence of the coatingafter his-tag binding did not influence the plasmonic peak of the

gold at 540 nm, which demonstrates the mild nature of the ligandbinding (Supplementary Fig. 7c). As shown in SupplementaryFig. 7d, only RGD-his Co-PoP-coated gold nanoparticles targetedU87 cells and free RGD inhibited the binding as determined bybackscatter microscopy. Co-PoP gold alone and 2H-PoP-coatedgold incubated with the RGD-his peptide were ineffective attargeting U87 cells.

Development of antigenic liposomes. Many of the monoclonalantibodies that broadly neutralize the human immunodeficiencyvirus (HIV) viral entry, such as 2F5, Z13 and 4E10, target aconserved linear epitope in the membrane proximal externalregion (MPER) of the gp41 envelope protein, which makes theMPER a prime target for HIV peptide vaccines40,41. However, it isexposed only during viral entry and attempts to use MPERpeptides to generate neutralizing antibodies have facedchallenges42,43. This has given rise to the paradigm thatvaccination strategies should consider antibody interaction withthe lipid bilayer in which the MPER is presented44. Recent effortshave made use of liposomes that contain the Toll-like receptor 4(TLR-4) agonist monophosphoryl lipid A (MPL) combined withliposome-bound MPER peptide sequences11,45,46. However, the useof a simple anchoring technique based on the binding of MPERhis-tagged polypeptides to Ni-NTA liposomes generated lowantibody titres47. We set out to examine if the same approachcould be enhanced with Co-PoP liposomes.

A liposomal-peptide vaccination system was used withthe MPER-his sequence NEQELLELDKWASLWNGGKGG-HHHHHHH. The MPER-his peptide was bound to Co-PoP

+–

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Figure 6 | HIV peptide vaccination using immunogenic Co-PoP liposomes. a, BALB/c or athymic nude mice were immunized with Co-PoP liposomes thatcontained 25 µg MPL and 25 µg his-tagged MPER peptide derived from the HIV gp41 envelope protein. The sera titres were assessed with ELISA using abiotinylated MPER peptide that lacked a his-tag and then probed with an anti-IgG secondary antibody. Arrows indicate the time of the vaccinations. Mean ± s.d.for n= 4 mice per group. b, Anti-MPER titre in BALB/c mice vaccinated as indicated. Mice were vaccinated on week 0 and week 2 and the serum wascollected on week 4. Mean ± s.d. for n= 4 mice per group. c, Sustained anti-MPER titre in mice vaccinated with Co-PoP liposomes that contained MPL relativeto Ni-NTA liposomes that contained MPL. Mean ± s.d. for n =4 mice per group. d, Neutralization of HIV infection in TZM-bl cells in the presence of theindicated antibodies. After vaccination using Co-PoP, IgGs were purified from pooled mouse sera using Protein G. The broadly neutralizing antibody 2F5 wasused as a control. Mean ± s.d. for n= 3.






liposomes that contained MPL. A single injection containing 25 µgMPER-his and 25 µg MPL was administered to BALB/c mice and toathymic nude mice. This elicited a titre on the order of 104 in boththe BALB/c mice and the nude mice, which demonstrates a stronghumoral immune response (Fig. 6a). This may be significantbecause HIV infects helper T cell populations, which makesresponses mediated by B cells important. After a booster injection,the anti-MPER titre in athymic nude mice was unaffected, but inhealthy mice there was a titre increase by an order of magnitude,which demonstrates a memory effect mediated by T cells. Thus,the vaccination protocol resulted in immunity mediated by bothB cells and T cells. Interestingly, a recent vaccination study usingMPL liposomes and a lipid-anchored Alzheimer’s-related peptidedemonstrated a response independent of T cells, without any boost-ing phenomenon13. That study used subcutaneous injections andlower MPL doses (12 versus 25 µg), which may account for thedifferent immune responses.

Next, various liposome and adjuvant components were exam-ined to better determine the specificity of the immune response(Fig. 6b). The MPER-his peptide did not elicit any antibodieswhen injected on its own, in Freund’s complete adjuvant or alongwith 2H-PoP liposomes that contained MPL. When the peptidewas administered with Co-PoP liposomes that lacked MPL, no anti-bodies were generated whatsoever. Another lipid adjuvant, trehalosedibehenate (TDB), also failed to elicit any antibody production.TDB does not act on TLR-4, which underlines the importance ofMPL in the immune activation of the liposomal vaccine system.When MPER-his and Ni-NTA liposomes were used, a weakantibody titre of less than 103 was achieved, consistent with previousreports47. However, when Co-PoP liposomes were used, a titre twoorders of magnitude greater was observed. Presumably, the stablebinding of the peptide to the liposomes in vivo was directly respon-sible for this effect. The Co-PoP immunization strategy was effec-tive, with antibody titres persisting for at least three months,whereas no antibodies were detected with Ni-NTA liposomes afterone month (Fig. 6c).

Post-vaccination sera from mice were pooled and purified usingProtein G agarose to yield purified immunoglobulin-G (IgG). Thiswas then used to assess the inhibition of viral entry by HIV (Fig. 6d).When the purified IgG from vaccinated mice was used at a final con-centration of 0.2 mg ml–1, viral entry was inhibited by more than75%. This efficacy of inhibition was greater than that of thebroadly neutralizing monoclonal antibody 2F5 when incubated ata concentration of 2 µg ml–1, but less than when incubated at20 µg ml–1. These data show the potential for a vaccinationapproach that makes use of Co-PoP liposomes with HIV-derivedpeptides to induce antibody generation that can prevent viral entry.

ConclusionCo-PoP and structures formed thereof represent simple and versatilematerials for binding his-tagged proteins and peptides. This coatingcan easily confer targeting or immunogenic properties to diversematerials, an approach that enables leverage of the his-taggedpolypeptides produced through modern peptide synthesis andgenetic engineering. These can be easily, reliably and stably insertedinto Co-PoP liposomes and other bilayer-coated materials for appli-cations in biological systems.

MaterialsSee the Supplementary Information for full details of the methods.

Polypeptides. Custom peptides were obtained from commercial sources (seeSupplementary Table 1). The recombinant heptahistidine-tagged Cerulean–Venusfusion protein was produced as previously described48.

PoP liposomes. 2H-PoP was synthesized as previously described23. The metallo-PoPs were generated by incubating excess metal acetates with PoP in methanol ortetrahydrofuran. Completion of the reaction was monitored by thin-layer

chromatography. The solvent was then removed by rotary evaporation and PoP wasextracted thrice with a chloroform:methanol:water mixture. The identity wasconfirmed by mass spectrometry. PoP liposomes were created using the thin-filmmethod and extruded through 100 nm membranes using a handheld extruder.Stoichiometry approximations were based on the assumption that each ∼100 nmliposome contained 80,000 lipids. For protein- and peptide-binding analyses,liposomes were formed with 10 mol% PoP along with 85 mol% DOPC (AvantiNo. 850375P) and 5 mol% PEG-lipid (Avanti No. 880120P). Ni-NTA liposomesincluded 10 mol% Ni-NTA lipid dioleoylglycero-Ni-NTA (Avanti No. 790404P) aswell as 10 mol% 2H-PoP. Sulforhodamine B (VWR No. 89139-502) liposomescontained 10 mol% PoP, 35 mol% cholesterol (Avanti No. 700000P), 55 mol%DOPC and PEG-lipid as indicated. For bilayer integrity, cell binding and in vivostudies, a 50 mM dye was used for lipid film hydration, whereas for microscopystudies a 10 mM dye was used. Free dye was removed by gel filtration.

Polypeptide binding. The fluorescent reporter protein (1 μg) was incubated with20 μg liposomes in 200 μl PBS. Fluorescence in the FRET channel was measured anddata were normalized to the FRET signal of the protein without the addition ofliposomes. EMSA experiments were performed using 2.5 μg protein incubated with50 μg liposomes. For imidazole displacement experiments, 1 μg reporter protein wasbound to 20 μg liposomes. Imidazole was then titrated and the binding was assessedwith fluorescence. For serum stability, 1 μg reporter protein was bound to 20 μgliposomes in 100 μl PBS and then an equal volume of FBS was added and thebinding was monitored by fluorescence. Peptide binding was assessed with RGD-hisFAM fluorophore quenching after incubation of 500 ng peptide with20 μg liposomes.

Targeting experiments. U-87 cells (2 × 104) and MCF-7 cells (2 × 104) were seededovernight in a 96-well plate. RGD-his peptide (500 ng) was bound with 20 μgliposomes loaded with sulforhodamine B, and then incubated with the cells for twohours. The media was removed, cells were washed and liposomal uptake was assessedby the fluorescence of sulforhodamine B after lysis with a 1% Triton X-100 solution.For confocal imaging, 104 cells were seeded overnight in a chamber slide and 20 μgliposomes were then added to the serum-containing media and incubated for twohours. The media was removed and the cells were washed prior to confocalmicroscopy. Animal procedures were conducted in accordance with the policies ofthe University at Buffalo Institutional Animal Care and Use Committee. Five-week-old female athymic nude mice that bore U87 flank tumours (∼5 mm) wereintravenously injected with 200 µl liposomes loaded with sulforhodamine B with orwithout cRGD-his. After 45 minutes, the mice were sacrificed and the organs wereextracted, homogenized in a 0.2% Triton X-100 solution and the fluorescenceassessed to determine the biodistribution.

Vaccinations. On days 0 and 14, eight-week-old female BALB/c mice (HarlanLaboratories) received hind ventral footpad injections that contained 25 μg MPERpeptide in 50 μl sterile PBS. Where indicated, the injections also included 25 μg MPLincorporated into the liposomes (Avanti No. 699800P). The anti-MPER titre wasassessed by enzyme-linked immunosorbent assay (ELISA) in 96-well streptavidin-coated plates (GBiosciences No. 130804). His-tag-free MPER biotin (1 μg) in 100 μlPBS that contained 0.1% Tween-20 (PBS-T) was incubated in the wells for two hoursat 37 °C. Wells were washed and mouse sera were diluted in PBS that contained 0.1%casein and then incubated. Wells were then washed with PBS-T and goat anti-mouseIgG-HRP (GenScript No. A00160) was added. The wells were washed again withPBS-T before the addition of tetramethylbenzidine (Amresco No. J644). Titres weredefined as the reciprocal serum dilution at which the absorbance at 450 nm exceededthe background by more than 0.05 absorbance units. Every sample was averagedfrom duplicate measurements.

Viral entry experiments. Viral entry experiments were carried out as previouslydescribed49. Sera from three mice immunized with MPER and Co-PoP liposomeswere pooled and IgG was isolated using immobilized Protein G beads (VWRNo. PI20398) according to manufacturer protocol. 2F5 was provided by the free NIHAIDS Reagent Program. Receptor cells (1 × 104 TZM-bl per well) were plated in a96-well plate the day before infection. HIV (multiplicity of infection of 0.1) wasincubated with antibodies for 30 minutes at 37 °C, added to the cells andspinoculated at 1,000g for one hour at 25 °C followed by a further incubation for twodays at 37 °C. Cell viability and viral entry were assessed, respectively, using theCellTiter-Fluor (Promega) and One-Glo (Promega) assays according tomanufacturer protocol.

Received 16 September 2014; accepted 16 March 2015;published online 20 April 2015

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AcknowledgementsThe authors thank J. R. Morrow and R. B. Bankert for valuable discussions, A. Siegel forassistance with confocal microscopy and the NIH AIDS Reagent Program. This work wassupported by grants from the National Institutes of Health (R01EB017270, DP5OD017898and HL77258).

Author contributionsS.G. and S.N. designed and produced the his-tagged fluorescence protein reporter. S.S.performed the synthesis, chemical characterization, binding, targeting and immunizationexperiments. J.G. led the animal experiments. A.J. and H.A.Y. carried out the HIV entryinhibition experiments. S.S. and J.F.L. conceived the project, interpreted the data and wrotethe manuscript.

Additional informationSupplementary information is available in the online version of the paper. Reprints andpermissions information is available online at www.nature.com/reprints. Correspondence andrequests for materials should be addressed to J.F.L.

Competing financial interestsThe authors declare no competing financial interests.





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